Kinetic study of NADPH activation using ubiquinone-rhodol fluorescent probe and an IrIII-complex promoter at the cell interior

Nicotine adenine dinucleotide derivatives NADH and NADPH are intimately involved in energy and electron transport within cells. The fluorescent ubiquinone-rhodol (Q-Rh) probe is used for NADPH activation monitoring. Q-Rh reacts with NADPH yielding its quenched hydroquinone-rhodol (H2Q-Rh) form with concurrent NADPH activation (i.e. NADP+ formation). NADPH activation can be enhanced by the addition of an IrIII-complex (i.e. [(η5-C5Me5)Ir(phen)(H2O)]2+) as a promoter. The rate of the Q-Rh fluorescence quenching process is proportional to the NADPH activation rate, which can be used to monitor NADPH. Experiments were performed in phosphate-buffered saline (PBS) solution and on HeLa cell cultures to analyze the kinetics of Q-Rh reduction and the influence of the IrIII-complex promoter on the activation of NADPH (in PBS) and of other intracellular reducing agents (in HeLa cells). There is a substantial increase in Q-Rh reduction rate inside HeLa cells especially after the addition of IrIII-complex promoter. This increase is partly due to a leakage process (caused by IrIII-complex-induced downstream processes which result in cell membrane disintegration) but also involves the nonspecific activation of other intracellular reducing agents, including NADH, FADH2, FMNH2 or GSH. In the presence only of Q-Rh, the activation rate of intracellular reducing agents is 2 to 8 times faster in HeLa cells than in PBS solution. When both Q-Rh and IrIII-complex are present, the rate of the IrIII-complex catalyzed reduction reaction is 7 to 23 times more rapid in HeLa cells. Concentration- and time-dependent fluorescence attenuation of Q-Rh with third-order reaction kinetics (reasonably approximated as pseudo-first-order in Q-Rh) has been observed and modelled. This reaction and its kinetics present an example of “bioparallel chemistry”, where the activation of a molecule can trigger a unique chemical process. This approach stands in contrast to the conventional concept of “bioorthogonal chemistry”, which refers to chemical reactions that occur without disrupting native biological processes.


Introduction
][3][4][5] In contrast to biorthogonal chemistry, "bioparallel chemistry" is attributed to chemical reactions involving articial molecules interacting with native biological processes, and has been introduced by Komatsu et al. in 2014. 6The intracellular activation of acetyl coenzyme A (acetyl-CoA) by tributylphosphine (PBu 3 ), and its uorescent detection, is considered the rst successful example of the bioparallel chemistry concept. 7In a further study, an articial reaction promoter (PBu 3 ) was used to control ATP concentration and acetylation of mitochondrial proteins. 8These results effectively illustrate that novel articial reaction promoters can be excellent candidates for intracellular imaging and are promising for the modulation of cellular functions.In 2013, Sadler and coworkers reported the reduction of quinone by reduced coenzyme NADH involving a cyclopentadienyl-Ir III catalyst complex in aqueous media. 9,10Subsequently, in 2014, Komatsu et al. reported the use of a uorescent ubiquinone-rhodol (Q-Rh) conjugate containing a biocompatible rhodol uorophore 11 for intracellular activation and imaging of nicotinamide adenine dinucleotide (NAD) derivatives NADH and NADPH.Both NADH and NADPH act as electron transporters in living cells and play a crucial role in metabolism. 12,13Here, a kinetic study has been undertaken to understand the reaction mechanism of NADPH in the presence of Q-Rh uorescent dye and an Ir III -complex (i.e.[(h 5 -C 5 Me 5 ) Ir(phen)(H 2 O)] 2+ ) promoter (Fig. 1a).Information regarding rates of chemical reactions (including in the biological system), reaction order, and rate-determining steps 14,15 is essential for the further development of quinone reduction processes that mimic the action of reductases, such as NADH ubiquinone oxidoreductase, 16 NADH cytochrome-b 5 , 17 and NADPH cytochrome P-450 reductase. 18n this study, we present an analysis of the Ir III -complex catalyzed reduction of Q-Rh by NADPH yielding hydroquinonerhodol (H 2 Q-Rh) and activated NADP + (Fig. 1a).This reaction is important as an one of the leading examples of bioparallel chemical processes in living organisms.

Kinetics in phosphate-buffered saline (PBS) solution
The Q-Rh uorescent probe (synthesized according to Komatsu et al. 6 ) has absorbance and uorescence maxima at 492 and 518 nm, respectively (Fig. 1b), with a uorescence quantum yield of 0.73 in phosphate-buffered saline (PBS) at pH = 7.4.The typical uorescence lifetime of rhodol-type dyes is around 4 ns in aqueous phosphate buffer. 19The reduced form of Q-Rh (i.e.H 2 Q-Rh) obtained using sodium dithionite (Na 2 S 2 O 4 ) 20 has signicantly attenuated UV-vis absorption and its uorescence emission is strongly quenched involving photoinduced-electron transfer (PET) mechanisms, as shown in Fig. 1b.Micrographs illustrating the time dependence of uorescence emission of Q-Rh at the interior of HeLa cells following addition of the Ir IIIcomplex are shown in Fig. 1c.Also in this study, we elucidate the kinetics behind the time dependence of uorescence emission intensity.However, rst we will consider the normal kinetics of Q-Rh to H 2 Q-Rh conversion under simple conditions (in PBS, pH = 7.4) in the presence of NADPH and in the absence/presence of the Ir III -complex promotor (which activates NADPH).
The following equation describes the reduction reaction of Q-Rh by NADPH.
It should be noted that the overall reaction in eqn (1) consists of the following two processes.
The reaction in eqn (2) describes the direct hydride transfer leading to the formation of hydroquinone anion (HQ − -Rh) and is followed by the reaction in eqn (3), where hydroquinone H 2 Q-Rh is formed due to the rapid protonation of HQ − -Rh by H + from the medium. 21This indicates that the reaction shown in eqn ( 2) is the rate-limiting step.Therefore, the reaction rate constant, k, for the overall reaction (eqn (1)) is the same as that for the reaction in eqn (2).
In the presence of the Ir III -complex reaction promoter, the following reversible association process between NADPH and Ir III -complex is assumed to occur.
where Ir represents the promoter Ir III -complex, NADPH$Ir is the complexed form of NADPH reactant with the Ir III -complex, and ) is the equilibrium association constant (square brackets denote concentrations of the species).Kinetics of the reaction in the presence of reactantpromoter NADPH$Ir complex is governed by the following reaction.
where k Ir is the reaction rate constant of this process (i.e. the process in which the NADPH$Ir complex reduces Q-Rh).Thus, in the presence of Ir III -complex promoter, all three processes described by the reactions in eqn (1), ( 4) and ( 5) are simultaneous.The solution of these kinetics is done using the following approach.The reaction progress was monitored by uorescence emission from the Q-Rh probe (at 518 nm).The reactions shown in eqn (1) and ( 5) lead to the following differential rate equation for the decrease of Q-Rh concentration.
Substituting the [NADPH$Ir] term using the denition of K Ir (given in the context of the reaction in eqn (4)) followed by rearrangement yields a differential rate equation for Q-Rh in the following form.
In this study, the concentration of Q-Rh uorescent probe is always signicantly lower than those of the NADPH reactant and Ir III -complex promoter.Therefore, they can be assumed constant, i.e.
[NADPH] = [NADPH] 0 and [Ir] = [Ir] 0 , where [NADPH] 0 and [Ir] 0 are initial concentrations of NADPH and Ir III -complex promoter, respectively.This situation is denoted by '0' subscripts in eqn (7).The above assumptions reduce the initially third-order rate eqn (7) (i.e., rst-order in [Ir], [NADPH] and [Q-Rh]) to pseudo-rst-order in Q-Rh concentration with a pseudo-rst-order rate constant The eqn (7) can then be solved analytically in the form of eqn (9). 14 where [Q-Rh] 0 is the initial concentration of Q-Rh probe.The time dependency of hydroquinone H 2 Q-Rh concentration can be readily derived considering the mass balance equation Taking into account that the uorescence emission of reacted quenched H 2 Q-Rh (at 518 nm) is 1/30 (=q) of the Q-Rh starting uorescence intensity (i.e.ca.97% quenching efficiency) due to the operation of PET mechanism (see Fig. 1b and Experimental section for more details), we can assume that the time dependence of the normalized uorescence intensity Then the resulting I n (t) can be expressed as eqn (11), where normalization means that I n (t = 0) = 1.
In order to extract the kinetic parameters, the timedependent normalized uorescence of Q-Rh (0.01 mM) with NADPH (1 mM) was measured at different Ir III -complex promoter concentrations in PBS solution, as shown in Fig. 2a.In the absence of Ir III -complex, the pseudo-rst-order rate constant reduces to k ′ = k[NADPH] 0 , and tting of eqn (11) to experimental data (Fig. 2a) yields a value of the reaction rate constant k = 0.28 ± 0.06 M −1 s −1 describing the kinetics in eqn (1).Aer the addition of Ir III -complex (0.5 and 1 mM) into the solution (and using already-known k), the tting procedure (Fig. 2a) further yields the value of the product k Ir K Ir = 764 ± 85 M −2 s −1 contained in the unreduced pseudo-rst-order rate constant k ′ in eqn (8).Table 1 summarises the kinetic parameters obtained.It can be seen that the presence of a small quantity of Ir III -complex promoter (for example, 1 mM) enhances the rate of the Q-Rh reduction reaction by a factor of 4; i.e. the promoting effect of Ir III -complex (1 mM) expressed as "enhancement ratio" is r enh ¼ k 0 with-Ir =k 0 without-Ir z 3:9.Note that the enhancement ratio is independent of NADPH concentration and has the general formula shown in eqn (12).
The enhancement ratio (plotted in Fig. 2b) can be used to determine how many times (i.e.r enh -times) faster is the Q-Rh reduction reaction in the presence of Ir III -complex promoter ([Ir] 0 in the units of M) compared to the uncatalyzed (unpromoted) reaction in eqn (1).The individual constants in the product k Ir K Ir cannot be extracted from the available kinetic  (11) with k and k Ir K Ir values from Table 1 and [NADPH] 0 as an independent variable.data, although this does not preclude further analysis of the intracellular uorescence behaviour of Q-Rh.It is clear that the non-zero K Ir constant (i.e.NADPH$Ir complex formation in eqn ( 4)) plays a substantial role in the overall Q-Rh reduction kinetics.This is also emphasized by the relatively large value of the catalytic "boost" factor ðk Ir K Ir Þ k ¼ 2736 AE 430 M À1 in eqn (12), which highlights the activity of the Ir III -complex.The pseudo-rst-order kinetics represented in eqn ( 7) was further tested by experiments where the initial concentration of NADPH was varied (at constant [Q-Rh] 0 = 0.01 mM and [Ir] 0 = 0.5 mM), with Q-Rh uorescence emission monitored over time aer the addition of Q-Rh to the solution at t = 0 min (Fig. 2c).The solid lines represent calculated (not tted) behaviour as obtained using eqn (7) with k and k Ir K Ir values taken from Table 1, [Q-Rh] 0 = 0.01 mM, [NADPH] 0 as a parameter (0-10 mM) and t as the independent variable.Data from Fig. 2c can also be used to generate the Q-Rh uorescence decay as a function of NADPH concentration at a given constant time of 0-10 min (Fig. 2d).The solid lines again represent calculated (not tted) behaviour using eqn (7) (with k and k Ir K Ir from Table 1), [Q-Rh] 0 = 0.01 mM, t as a parameter (0-10 min) and [NADPH] 0 as the independent variable.It can be seen that the presented kinetic model describes well the experimental data.
Overall, from the above analyses, the kinetic model as introduced in eqn ( 1), ( 4) and ( 5), with the analytical solution represented by eqn ( 9) and ( 8) together with values in Table 1, yields a good description of the Q-Rh reduction process (i.e.formation of H 2 Q-Rh) and NADPH activation process (i.e.formation of NADP + ) in the presence of Ir III -complex promoter in PBS solution (at pH = 7.4).

Kinetics at the interiors of HeLa cells
A series of experiments were performed using HeLa cells in order to analyze the intracellular kinetics of Q-Rh reduction with simultaneous NADPH activation.HeLa cells (with cellular passage number in the range 5-10) were incubated (30 min., 37 °C) with Q-Rh (0.01 mM) in Hanks' balanced salt solutions (HBSS) at pH = 7.4.Prior to uorescence observation, HeLa cells placed in a glass bottom dish were rinsed twice with HBSS, and then the dish was lled with fresh HBSS.The average uorescence intensity of each individual cell of the sample was obtained using a confocal laser scanning microscope on a cell culture maintained at 25 °C.Micrographs of Q-Rh uorescence quenching in HeLa cells prior to and following the addition of Ir III -complex ([Ir] 0 = 0.1 mM) are shown in Fig. 3a.Time dependence of the average uorescence intensity as obtained from individual cells is plotted in Fig. 3b (for [Ir] 0 = 0.1 mM) and Fig. 3c (for [Ir] 0 = 0.5 mM).The sudden increase in uorescence intensity following the addition of Ir III -complex is most likely due to injection shock (to some extent, an effect similar to addition of hypertonic solution and the corresponding change in the osmotic pressure).For further analyses, it is convenient to average and normalize the data before and aer the addition of Ir III -complex, as shown in Fig. 3d and e (black lines).We have also observed that upon addition of Ir III -complex into the HeLa cell culture (in the absence of Q-Rh), the subsequent NADPH activation (or activation of other species) causes cell membrane disintegration, which is probably caused by the intracellular downstream signalling cascade (which regulates cell growth, proliferation, differentiation, and it can also trigger cell apoptosis 22 ).We have used calcein acetoxymethyl (calcein-AM), a green uorescent dye useful for cell viability monitoring, to stain the cytosol and then observed the dye leakage through HeLa cell membranes following the addition of Ir III -complex, as shown in Fig. 3d and e (orange lines) for addition of 0.1 mM and 0.5 mM of Ir III -complex, respectively.Due to its considerable effect, the leakage rate (k leak ) has to be included in the overall Q- Data are normalized prior to and following the addition of Ir III -complex (at t = 0 min).Orange lines show averaged and normalized fluorescence data of calcein stained (0.01 mM) HeLa cells after the addition (at t = 0 min) of (d) 0.1 mM and (e) 0.5 mM of Ir III -complex depicting the HeLa cell membrane disintegration (note that in this case, the Q-Rh is not present in HeLa cells).Grey and light orange backgrounds correspond to standard deviations.Blue and red lines are fitted according to eqn ( 14) and ( 16 Rh reduction kinetics in HeLa cells.The leakage process was modelled as a release process in a conned space (i.e. the glass bottom dish), which is governed by the following differential equation.
where [Cal] is the time-dependent concentration of calcein in the HeLa cells and [Cal] eq (s0) is the equilibrium calcein concentration at innite time introduced due to conned space.The solution of eqn ( 13) can be expressed in terms of normalized intensity (similarly as for eqn (11)) as where p = [Cal] eq /[Cal] 0 is the fraction of remaining calcein uorescence due to the conned space, and [Cal] 0 is the initial calcein concentration at t = t 0 .Normalization means that I n,Cal (t = t 0 ) = 1, where t 0 can be interpreted as a lag time in uorescence decrease aer the addition of Ir III -complex in the HeLa cell culture.Eqn ( 14) is then tted into experimentally observed calcein uorescence data, as shown in Fig. 3d and e (blue lines; values of tted parameters are shown in the caption).The leakage rate (k leak ) has a power law type dependence on added Ir III -complex concentration k leak = 0.216[Ir] 0 1/2 , as shown in Fig. 4a (and the inset).
Using the above analysis of the HeLa cell membrane leakage rate aer the addition of Ir III -complex, we can construct the overall pseudo-rst-order rate constant for Q-Rh reduction kinetics inside the HeLa cells in the following form: where k leak can be well approximated as k leak = 0.216[Ir] 0 1/2 (see Fig. 4a).[27][28] The a eff cell and b eff cell coefficients express effective rate increase of uncatalyzed Q-Rh reduction (shown in eqn ( 1)) and Ir IIIcomplex catalyzed reduction (shown in eqn ( 5)), respectively, in HeLa cells as compared to the experiments in PBS solution (where of a eff cell = b eff cell = 1).It can also be noted that the a eff cell and b eff cell coefficients refer to the increase of Q-Rh reduction rate for t < 0 min and t > 0 min, respectively, as shown in Fig. 3d and e.
The rate constant k 0 cell is then used in eqn ( 16) (which is derived from eqn (7) analogously as eqn (11)) and tted into normalized and averaged experimental data, as shown in Fig. 3d and e (red lines).
The t 0 is a lag time in Q-Rh uorescence decrease aer the addition of Ir III -complex.In eqn ( 16) a eff cell , b eff cell and t 0 are tted parameters.0][31][32][33] Therefore, the tting of the experimental data in Fig. 3d and e (red lines) has been performed for various NADPH concentrations, and a eff cell and b eff cell coefficients were obtained as a function of [NADPH] 0 (Fig. 4b).The data in Fig. 4b indicate that in the HeLa cell available NADPH concentration range (grey zone), the coefficients are within the following ranges: 2 < a eff cell < 8 and 7 < b eff cell < 23.From the tting procedure, it can also be noted that in equilibrium, the Ir III -complex uorescence quenching efficiency on Q-Rh seems to be the same (within the experimental error) as in PBS solution, i.e. 97%.This large increase in reaction rates is due to the presence of the abovementioned reducing species in the cytosol, which apparently contribute to the Q-Rh reduction process before and, even more signicantly, aer the addition of Ir III -complex promoter.This can be quantied by the "enhancement ratio" r enh,cell , from which the component of leakage process has been removed, as The ratio b eff cell /a eff cell z 2.8 over the whole NADPH admissible concentration region (Fig. 4b), which indicates an about 2.8 times higher Ir III -complex activity for the Q-Rh reduction process in HeLa cells over that in PBS solution (as seen by comparison with eqn ( 12)).Since the values of a eff cell and b eff cell are larger than 1 in the whole admissible region, it also suggests that other intracellular reducing agents besides NADPH (e.g.NADH, FADH 2 , FMNH 2 or GSH) are also activated by the presence of the Q-Rh uorescence probe alone (2 < a eff cell < 8) and more strongly by the further addition of Ir III -complex (7 < b eff cell < 23).It is worth mentioning that GSH is the main agent in the reduction process of intracellular quinones. 286][27][28] The high values of a eff cell and b eff cell coefficients (and accordingly increased k 0 cell ) most likely reect this situation.5][36][37][38] Lower pH (less than 5.7) increases the reaction rate k in eqn (1). 39,40However, other research 21 has shown that there is almost no effect on the reaction rate k in the pH range from 6 to 8. Therefore, there might be a limited contribution to the increase of the overall Q-Rh reduction rate constant in HeLa cells ðk 0 cell Þ, which can be assigned to lower intracellular pH.
These results imply that the Q-Rh reduction process, which is accompanied by uorescence quenching, can be used for the estimation of the relative rate of NADPH activation (i.e. the rate of NADP + formation).The addition of Ir III -complex promoter can further increase this activation rate.In PBS solution, this process can be well controlled with an enhancement factor due to the Ir III -complex concentration expressed in eqn (12).In HeLa cells, the kinetics can also be analyzed, although the Q-Rh uorescence quenching rate should be rather interpreted as the relative rate of activation involving several other intracellular reducing agents (not only NADPH).The presence of Ir IIIcomplex also has a profound effect on the of the activation process, as expressed in eqn (17).Moreover, this activation caused by Ir III -complex inside the HeLa cells initiates a downstream signalling cascade, which results in cell membrane disintegration and cell death.This cascade is perhaps caused by the generation of reactive oxygen species, such as H 2 O 2 , which has been reported for similar organoiridium complex systems. 10,41,42The intrinsic cytotoxicity of the Ir III -complex is also evident from monitoring of calcein dye uorescence aer the addition of Ir III -complex (in the absence of Q-Rh), which initiates the cytosol leakage and eventual cell death.On the other hand, HeLa cells incubated only with Q-Rh (without the addition of Ir III -complex promoter) showed no signs of decreased cell viability.

Conclusions
We have shown that in the controlled environment of PBS solution, the uorescent ubiquinone-rhodol (Q-Rh) probe reacts with NADPH leading to its quenched hydroquinone-rhodol (H 2 Q-Rh) form with simultaneous NADPH activation.This activation can be further increased by the addition of Ir IIIcomplex (i.e.[(h 5 -C 5 Me 5 )Ir(phen)(H 2 O)] 2+ ) promoter.The rate of Q-Rh uorescence quenching process is proportional to NADPH activation rate.The kinetics of this process can be wellmodelled by rst-order kinetics for Q-Rh concentration with the pseudo-rst-order rate constant involving the concentrations of Ir III -complex and NADPH.
Furthermore, we performed experiments on HeLa cells to analyze the intracellular kinetics of Q-Rh reduction and the inuence of Ir III -complex promoter on the activation of intracellular reducing agents.We found that this process can also be modelled by modied rst-order kinetics for Q-Rh.However, the Ir III -complex stimulates downstream intracellular processes, which result in HeLa cell membrane disintegration and leakage of the cytosol.Our kinetic model accounts for this process.Therefore, the actual uorescence quenching of Q-Rh caused by reduction reactions can be quantied and their kinetic parameters extracted.There is a substantial increase in the Q-Rh reduction rate (accompanied by a corresponding increase of uorescence quenching) inside the HeLa cells, especially aer the addition of Ir III -complex promoter.This increase is partially due to the leakage process but also due to the nonspecic activation of other intracellular reducing agents other than NADPH, such as NADH, FADH 2 , FMNH 2 or GSH (which might have the dominant contribution due to high intracellular GSH concentrations at mM levels).In the presence only of Q-Rh, the activation rate of the intracellular reducing agents is about 2 to 8 times greater in HeLa cells than in PBS solution.In the presence of both Q-Rh and Ir III -complex, the Ir III -complex catalyzed reduction reaction is about 7 to 23 times faster in HeLa cells.
The activation of NADPH or other intracellular species with simultaneous monitoring of this process can be used to exploit unique chemical reactions.This concept stands in contrast to the conventional, widely recognized concept of bioorthogonal chemistry.We have coined the term "bioparallel chemistry" to differentiate this approach.The analyses of Ir III -complex promoted NADPH activation, and its monitoring by Q-Rh uorescence probe given in this study represents the rst attempt to analyze the kinetics of a bioparallel reaction at the interiors of cells.

General
Fluorescence spectra were measured on a JASCO FP-8500 spectrouorophotometer using a quartz cuvette with a 1 cm path length.Phosphate-buffered saline (100 mM, pH 7.4) was used as a solvent.HeLa cells were obtained from RIKEN (Tsukuba, Japan), and cultured in Dulbecco's Modied Eagle Medium (DMEM) (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS), 50 U mL −1 of penicillin and 50 mg mL −1 streptomycin at 37 °C under a humidied atmosphere of 5% CO 2 .
4.2 Time-dependent uorescence measurements in PBS (Ir III -complex concentration dependence) Time-dependent uorescence intensity of Q-Rh (0.01 mM) reduction in PBS solution (at pH 7.4) was measured in the absence of the promoter (blank measurement), and with 0.5 mM and 1 mM of the Ir III -complex promoter, and with 1 mM NADPH which was added at time t = 0 min.During the measurements (with excitation wavelength l ex = 488 nm), the uorescence was observed at its maximum at l em = 518 nm.Quenched uorescence of Q-Rh reduced by sodium dithionite (Na 2 S 2 O 4 ) 20 to H 2 Q-Rh in PBS buffer was lowered to 1/30 of its original value (at 518 nm). 63 Time-dependent uorescence measurements in PBS (NADPH concentration dependence) Time-dependence of uorescence intensity during Q-Rh (0.01 mM) reduction was measured in PBS solution (at pH 7.4) with 0.5 mM of the Ir III -complex promoter following the addition (at t = 0 min) of various concentrations of NADPH (0, 0.25, 0.5, 1, 2.5, 5, and 10 mM).For these measurements, the excitation wavelength was l ex = 488 nm, and the uorescence maximum was observed at l em = 519 nm.

Time-dependent uorescence imaging of Q-Rh in HeLa cells
For uorescent imaging experiments, a confocal laser scanning microscope system (FluoView FV1000; Olympus, Tokyo, Japan) mounted on an inverted microscope (IX81; Olympus) with a 40× or 60× oil-immersed objective lens was used.The uorescence imaging measurements were performed on HeLa cells cultured on glass-bottomed dishes (Iwaki, Tokyo, Japan).For Q-Rh dye loading, the cells were incubated for 30 min at 37 °C in Hanks' balanced salt solutions (HBSS) containing NaCl (137 mM), KCl (5.4 mM), CaCl 2 (1.3 mM), MgCl 2 (0.5 mM), MgSO 4 (0.4 mM), Na 2 HPO 4 (0.3 mM), KH 2 PO 4 , (0.4 mM), NaHCO 3 (4.2 mM), D-glucose (5.6 mM), HEPES (5.0 mM) (pH was adjusted to 7.4 using NaOH) in the presence of 0.01 mM Q-Rh.HeLa cells were washed twice with HBSS solution to remove the remaining extracellular dye, and uorescence imaging measurements were subsequently performed.Q-Rh was excited at l ex = 488 nm, with the signal being observed at 500-600 nm.Fluorescence images were acquired and processed in the FluoView soware package (Olympus).The uorescence intensities were determined by calculating the average intensity within a dened region of interest that encompassed the cell body of each cell.Fluorescence imaging indicates that Q-Rh is uniformly distributed in the cytosol with partial accumulation in mitochondria.

Fig. 2
Fig. 2 (a) Plot of Q-Rh time-dependent normalized fluorescence intensity I n (t) (at 520 nm) as a function of Ir III -complex promoter concentration: [Ir] 0 = 0 mM (red data points), 0.5 mM (blue data points), and 1 mM (green data points).Experiments were performed in PBS solution (100 mM, pH = 7.4, 25 °C) at constant [NADPH] 0 = 1 mM and [Q-Rh] 0 = 0.01 mM.Solid lines are the best fits using eqn (11).(b) Plot of enhancement ratio r enh as a function of Ir III -complex promoter concentration.The data points are obtained from the evaluation of r enh independently from each experiment in (a).The grey solid line is a plot of eqn (12).(c) Plot of time-dependent Q-Rh normalized fluorescent intensity I n (t) (at 520 nm) recorded after Q-Rh addition ([Q-Rh] 0 = 0.01 mM) at t = 0 min into the PBS solutions (100 mM, pH = 7.4, 25 °C) containing constant Ir III -complex concentration ([Ir] 0 = 0.5 mM) and varying NADPH concentration ([NADPH] 0 = 0-10 mM).Solid lines are plots (not fits) of eqn (11) with k and k Ir K Ir values from Table 1 and t is an independent variable.(d) Plot of Q-Rh normalized fluorescent intensity I n ([NADPH] 0 ) (at 520 nm) as a function of [NADPH] 0 concentration recorded at various times (t = 0-10 min) after Q-Rh addition ([Q-Rh] 0 = 0.01 mM) into the PBS solutions (100 mM, pH = 7.4, 25 °C) containing constant Ir III -complex concentration ([Ir] 0 = 0.5 mM).Solid lines are plots (not fits) of eqn(11) with k and k Ir K Ir values from Table1and [NADPH] 0 as an independent variable.

Fig. 3
Fig. 3 (a) Micrographs of fluorescence quenching of Q-Rh stained (0.01 mM) HeLa cells after addition of Ir III -complex (0.1 mM) at 25 °C.Normalized fluorescence time profiles from individual cells are shown in (b) and (c), and averaged data are shown in (d) and (e).(b) and (c) Plots of normalized fluorescence time profiles as obtained from individual HeLa cells (l ex = 488 nm, observed at 500-600 nm, 25 °C) stained with Q-Rh (0.01 mM) after the addition (at t = 0 min) of (b) 0.1 mM and (c) 0.5 mM of Ir III -complex.(d) and (e) Black lines show averaged and normalized Q-Rh fluorescence data from (b) and (c).Data are normalized prior to and following the addition of Ir III -complex (at t = 0 min).Orange lines show averaged and normalized fluorescence data of calcein stained (0.01 mM) HeLa cells after the addition (at t = 0 min) of (d) 0.1 mM and (e) 0.5 mM of Ir III -complex depicting the HeLa cell membrane disintegration (note that in this case, the Q-Rh is not present in HeLa cells).Grey and light orange backgrounds correspond to standard deviations.Blue and red lines are fitted according to eqn (14) and (16), respectively.Calcein data fitted parameters: (d) k leak = (2.18 ± 0.34) × 10 −3 s −1 , p = 0.14 ± 0.03, t 0 = 1.5 min; (e) k leak = (4.82± 0.21) × 10 −3 s −1 , p = 0.10 ± 0.01, t 0 = 1.2 min.
Fig. 3 (a) Micrographs of fluorescence quenching of Q-Rh stained (0.01 mM) HeLa cells after addition of Ir III -complex (0.1 mM) at 25 °C.Normalized fluorescence time profiles from individual cells are shown in (b) and (c), and averaged data are shown in (d) and (e).(b) and (c) Plots of normalized fluorescence time profiles as obtained from individual HeLa cells (l ex = 488 nm, observed at 500-600 nm, 25 °C) stained with Q-Rh (0.01 mM) after the addition (at t = 0 min) of (b) 0.1 mM and (c) 0.5 mM of Ir III -complex.(d) and (e) Black lines show averaged and normalized Q-Rh fluorescence data from (b) and (c).Data are normalized prior to and following the addition of Ir III -complex (at t = 0 min).Orange lines show averaged and normalized fluorescence data of calcein stained (0.01 mM) HeLa cells after the addition (at t = 0 min) of (d) 0.1 mM and (e) 0.5 mM of Ir III -complex depicting the HeLa cell membrane disintegration (note that in this case, the Q-Rh is not present in HeLa cells).Grey and light orange backgrounds correspond to standard deviations.Blue and red lines are fitted according to eqn (14) and (16), respectively.Calcein data fitted parameters: (d) k leak = (2.18 ± 0.34) × 10 −3 s −1 , p = 0.14 ± 0.03, t 0 = 1.5 min; (e) k leak = (4.82± 0.21) × 10 −3 s −1 , p = 0.10 ± 0.01, t 0 = 1.2 min.

Fig. 4
Fig. 4 (a) Plot of leakage rate (k leak ) against the concentration of added Ir III -complex [Ir] 0 in HeLa cell cultures at 25 °C.The inset shows linear dependence of k leak on [Ir] 0 1/2 indicating a power law dependence.(b) Effective rate increase coefficients a eff cell and b eff cell for uncatalyzed and Ir III -complex catalyzed Q-Rh reduction in HeLa cells, respectively, as a function of intracellular NADPH concentration.The grey region denotes HeLa cell available NADPH concentration range.